1. Field of the Invention
The present invention relates to novel fluorescent dyes, quencher dyes and minor groove binders with enhanced polarity derived from arsonic acid conjugated thereto. The present invention further relates to methods of staining cells, labeling proteins and preparing oligonucleotide probes conjugated with polar arsonate dyes under the condition of automated synthesis.
2. Description of the Related Art
Dye-labeled synthetic oligonucleotides have been used for years as sequence-specific probes for complementary DNA and RNA targets. These methods have broad applications in forensics, molecular biology and medical diagnostics since they allow the identification and quantitation of specific nucleic acid targets. Early uses of DNA probes relied on radioactive labels (typically 32P), while recent methods use reporter molecules that include chemiluminescent and fluorescent groups. Improved instrumentation has allowed the sensitivity of these spectroscopic methods to approach or surpass the radio-labeled methods. Fluorescent dyes having high quantum yield are particularly suitable for biological applications in which a highly sensitive detection reagent is desirable.
Recently developed detection methods employ the process of fluorescence resonance energy transfer (FRET) for the detection of probe hybridization rather than direct detection of fluorescence intensity. In this type of assay, FRET occurs between a donor fluorophore (reporter) and an acceptor molecule (quencher) when the absorption spectrum of the quencher molecule overlaps with the emission spectrum of the donor fluorophore and the two molecules are in close proximity. The excited-state energy of the donor fluorophore is transferred to the neighboring acceptor by a resonance dipole-induced dipole interaction, which results in quenching of the donor fluorescence. The efficiency of the energy transfer between the donor and acceptor molecules is highly dependent on distance between the molecules. Other mechanisms of fluorescence quenching are also known, such as, collisional and charge transfer quenching.
Typically, detection methods based on FRET are designed in such a way that the donor fluorophore and acceptor molecules are in close proximity so that quenching of the donor fluorescence is efficient. During the assay, the donor and acceptor molecules are separated such that fluorescence occurs. FRET-based detection assays have been developed in the fields of nucleic acid hybridization and enzymology. Several forms of the FRET hybridization assays are reviewed (Nonisotopic DNA Probe Techniques, Academic Press, Inc., San Diego 1992, pp. 311-352). Quenching can also occur through non-FRET mechanisms, such as collisional quenching (see, Wei, et al., Anal. Chem. 66:1500-1506 (1994)).
An oligonucleotide probe comprising a reporter fluorophore and a quencher is typically non-fluorescent. Such an oligonucleotide probe is, however, capable of reporting the presence of a target sequence after the hybridization occurs by emitting detectable fluorescence. Because the hybridization or PCR process alters the conformation of the oligonucleotide probe or enzymatically cleaves the dye from the probe, the reporter fluorophore and the quencher become physically separated and the fluorescence is thereby restored. Assay formats using oligonucleotide probes which become fluorescent as DNA amplification occurs in “real time” PCR are described in details in Tyagi et al., Nat. Biotech., 16: 49-53 (1998) and Lee et al., Nucl. Acid Res., 21: 3761-3766 (1993). Fluorophore-oligonucleotide-quencher conjugates useful as DNA probes in real time PCR are further described in details in U.S. Pat. Nos. 6,727,356, 6,653,473 and 6,323,337.
Fluorescent dyes and quenchers, however, may substantially change the physical properties of nucleic acids probes due to their generally hydrophobic nature. This is a particularly difficult problem when the probe has more than one dye conjugated thereto or when the probe contains purine-rich sequences or other hydrophobic residues like minor groove binders or intercalators.
This problem has been partially resolved by introducing dyes containing several polar groups, usually sulfonic groups, to a probe. Sulfonic groups bring negative charge to the dye molecule, and the charge decreases the inherent tendency of molecules to form aggregates due to increased polarity imparted by the sulfonyl moieties [Mujumdar R. B., Ernst L. A., Mujumdar S. R., Lewis C. J., Waggoner A. S. (1993) Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. Bioconj. Chem. 4: 105-111]. Ring sulfonation also increases the brightness of many dyes such as Cy3, Cy5 [Wessendorf M. W., Brelje T. C. (1992) Which fluorophore is brightest? A comparison of staining obtained using fluorescein, tetramethylrhodamine, lissamine rhodamine, Texas Red and Cyanine 3.18. Histochemistry 98, pp 81-85] Cascade Blue [Whitaker J. E., Haugland R. P., Moore P. L., Hewitt P. C., Resse M., Haugland R. P. (1991) Cascade Blue derivatives: water soluble, reactive, blue emission dyes evaluated as fluorescent labels and tracers. Anal. Biochem. 198, pp 119-130] in aqueous media and remarkably increases photostability of rhodamine dyes [Panchuk-Voloshina N., Haugland R. P., Bishop-Stewart J., Bhalgat M. K., Millard P. J., Mao F., Leung W.-Y., Haugland R. P. (1999) Alexa Dyes, a Series of New Fluorescent Dyes that Yield Exceptionally Bright, Photostable Conjugates. J Histochem Cytochem 47(9), pp 1179-1188]. Some of these dyes are available commercially from Molecular Probes, Inc. and known under trademark Alexa Fluor® dyes. These water-soluble dyes are sold as activated succinimidyl esters and are popular labels for proteins and nucleic acids. Polar sulfonyl fluorescent dyes are described in U.S. Pat. Nos. 6,130,101, 5,268,486 and 6,399,392.
There remains a need in the art to provide polar dyes for biological staining, oligonucleotide probes and proteins conjugated to polar dyes with improved properties such as retaining high quantum yields and hydrophilicity, and to provide such dye-labeled probes by automated synthesis.